a) Field of the Invention
The present invention is directed to an adjustable coupling in and/or the detection of one or more wavelengths in a microscope. In particular, the invention is directed to such coupling and detection in the beam path of a confocal microscope.
b) Description of the Related Art
WO Patent 95/07447 (DE 4330347C2) describes a device which is arranged in the detection space of a laser scanning microscope. A main beam splitter, as it is called, (reference number 8 in FIG. 2) must be used in this arrangement.
When a plurality of lasers are used, this same main beam splitter is a very complicated optical layer system that reflects the laser light with only limited selectivity and effectiveness and, because of the non-optional level of edge steepness and reflectivity, the light emitted by the fluorochromes is reflected only with (high) losses.
In general, main beam splitters are those components of a laser scanning microscope which most limit the efficiency and selectivity.
U.S. Pat. No. 4,519,707 describes a multi-spectral detection system with dispersion and separate detection.
JP 493915 describes a spectroscopic system for remote sensing with a plurality of detector elements for wavelength-selective detection of the sensed object.
JP 61007426 describes a photometer with a fluorescence measurement filter in the dispersive light of the object.
DE 19510102 C1 describes a confocal fluorescence microscope for evaluation of fluorescent light with two prism spectrometers in the excitation light path, wherein the first prism spectrometer fans out the excitation light exiting by means of a first stripe diaphragm and the second prism spectrometer fans out the fluorescent light exiting from a second stripe diaphragm and a third prism spectrometer is provided in front of a detector.
Many locations on the object are illuminated and examined simultaneously. In so doing, the entire object plane is illuminated in parallel monochromatically. The achievable confocal effect and contrast depends in these arrangements on the coverage of the illuminated planes with transparent locations. In order to be able to make effective use of these arrangements, there must be a minimum coverage that limits the achievable contrast to 1:100 . . . 1:25. The use of slits leads to a textured confocal effect. This arrangement has disadvantages in terms of application, especially for multiple fluorescence.
The arrangement with three spectrometers which are connected one behind the other and with the use of selection elements in the form of gratings requires an enormous expenditure on adjustment and high stability of adjustment. The use of three duplicate prism spectrometers requires very close manufacturing tolerances. The use of slits for field illumination and the guiding of fluorescent light through the intermediate spaces either results in a considerable portion of the fluorescent light being lost when there is a high degree of coverage in that there is only a low permissible dispersion of the spectrometers or results in a low light yield or light efficiency in illumination at higher dispersions because the slits must be at a distance from one another corresponding at least to the spacing of the spectral width. Multiple fluorescences can be analyzed simultaneously in this arrangement only through increased expenditure.
It is the primary object of the invention to replace the conventional complicated main beam splitter with simpler components while at the same time improving flexibility, selectivity and efficiency, wherein the selected arrangement should also be suitable for spectral separation of fluorescent light returning from the object in a serially operating confocal laser scanning microscope with the highest requirements with respect to contrast and efficiency.
This is achieved in that the laser light which advantageously, but not compulsorily, emerges from the end of a fiber is sent through a spectrograph and a special band selector is arranged in the image plane thereof, that is, a comb with narrow fully-reflecting mirrors for fluorescence applications or with partially reflecting mirror stripes for reflection applications. The little mirrors are positioned at selected locations of the wavelengths of the laser or lasers and reflect the light of the desired wavelengths back into the spectrographs so as to be offset by a small angle. In so doing, all illuminating light advantageously returns to an individual point lying very close to a fiber for coupling in the laser light, the pinhole of the laser scanning microscope being located at this point.
Light which returns from the object with the wavelengths of the illumination impinges on the little mirrors of the band selector and, in the case of fluorescence, is fully reflected in the direction of the fiber and therefore effectively separated from the light to be detected; in the case of reflection, it passes partially through the partially reflecting little mirrors as in reflected-light microscopes and can be detected. This will be discussed more closely later on.
In the case of fluorescence, the returning light has wavelengths different than those of the illuminating light and therefore impinges in the image plane of the spectrograph in the neighborhood of the little mirrors.
Even wavelengths located next to the exciting wavelengths around the resolution of the spectrographs can be detected by the invention. These wavelengths are located much closer to the exciting wavelengths than would be possible in the case of dichroic splitters and losses are lower than with splitter layers because of the possible higher transmission. In this case, a plurality of regions of any width and spectral position can be cut out of the spectrum and supplied to different receivers by means of suitably shaped glass wedges such as those used in optical testing with spectrographs for demonstrating subtractive and additive color mixing.
A mirror unit comprises a transparent glass plate with small parallel mirrors at the locations of the anticipated laser wavelengths or at the locations of those laser wavelengths desired for examination, these laser wavelengths enter together, are dispersed, reflected at another location in the prism by the mirrors and imaged on the pinhole.
The mirrors can also be partially reflecting in order to enable detection (passage) of the illumination wavelength (reflection applications).
In fluorescence applications, the mirrors are fully reflecting in order to prevent the detection being influenced by the illumination wavelengths.
The specimen light (fluorescent light) is spectrally separated by at least one dispersive element, parallelized by a field lens and imaged through small glass wedges at different locations via a collector.
Without the wedges which cause a separation of the locations of impingement behind the collector, all of the beams would land in the focal point of the collector.
The spectral width (channel width) is changed by means of vertical wedge displacement, the spectral region of concern (channel position) is selected by horizontal wedge displacement.
Since the wedge angle is constant, there is no change in the deflection in the direction of the receiver.
The wedge angle determines the location of impingement and can be changed by exchanging wedges.
Further, prismatic lenses formed, for example, by lenses glued to wedges or decentered lenses formed by decentered lenses could be used. In this case, the detectors would have to be displaced along with the respective wedges when these lenses are displaced.
In one aspect of the present invention, a device for the adjustable detection of object light coming from an illuminated object, in a confocal microscope beam path, comprises:
at least one dispersive element for wavelength separation of the object light; and
means arranged in the wavelength-separated portion of the object light for the adjustable stopping down or cutting out of at least one wavelength region and deflection in the direction of at least one detector.
In another aspect of the present invention, a combination comprises:
a device comprising at least one dispersive element for wavelength separation of the illumination light; and at least one at least partially reflecting element arranged in the wavelength-separated portion of the illumination light for reflecting back at least one wavelength region in the direction of the microscope illumination;
with at least one of
a device comprising:
at least one dispersive element for wavelength separation of the object light; and means arranged in the wavelength-separated portion of the object light for the adjustable stopping down or cutting out at least one wavelength region and deflection in the direction of at least one detector;
and a device comprising:
at least one dispersive element for wavelength separation of the object light; and at least one prism-shaped wedge which is arranged in the wavelength-separated portion of the object light, is made of light-transparent and light-refracting material and whose position is adjustable vertical to the light direction in at least one direction;
wherein said combination is in a microscope.
In one embodiment, at least one dispersive element is used as a common element for coupling in the illumination light and for detecting the object light. In another embodiment, different dispersive elements are used for coupling in the illumination light and for detecting the object light.
The invention is described more fully hereinafter with reference to the schematic illustrations.